This project will investigate the molecular mechanisms which regulate expression of dihydrofolate reductase (EC 1.5.1.3) in S. cerevisiae (baker's yeast). This system offers an unusual opportunity to use the combined approaches of genetic manipulation and biochemical characterization in studying an eucaryotic gene. Techniques for selection, mapping, and genetic characterization of mutations in yeast are powerful and well described; dihydrofolate reductase ("FH2Rase") is a small monomeric protein easily assayed and purified. Most important, cell harboring mutations which cause either overproduction of FH2Rase or changes in the enzyme itself can be directly selected, because they are able to grow in the presence of inhibitory folic acid analogs. We will use this selection to obtain FH2Rase mutants of yeast, and then distinguish the enzyme overproducers from mutants affecting FH2Rase structure by assaying the enzyme in cell-free extracts. We will map these mutants and study their complementation and dominance. These experiments will provide a fine-structure map of the FH2Rase gene, define control loci regulating its expression, and determine map distances between the structural gene and its control elements. We will also purify FH2Rase from yeast by affinity chromatography, then characterize the tryptic peptides of the enzyme and determine their order within the polypeptide by straightforward protein chemistry. Mutations which map in the FH2Rase gene will be located in the polypeptide's sequence by finding which specific peptide is changed by each mutation. Aligning the resulting biochemical map of FH2Rase with a genetic map of the same mutations will tell us whether any nearby control locus is at the amine-terminal end of the gene, where it could not (but might code for a regulatory protein). Eventually biochemical studies on synthesis, processing, and translation of FH2Rase mRNA in normal and overproducer yeast will define the level (transcription or translation) at which FH2Rase expression is controlled.